Low-density Granulocytes Research Articles

Cardiovascular disease in systemic lupus erythematosus: an update.

The mechanisms leading to the development of premature atherosclerosis and vascular injury in systemic lupus erythematosus (SLE) remain to be fully elucidated. This is a comprehensive review of recent research developments related to the understanding of cardiovascular disease (CVD) in lupus. SLE patients with lupus nephritis display significantly increased risk of myocardial infarction and CVD mortality than SLE patients without lupus nephritis. SLE disease-related parameters could be taken into consideration when calculating CVD risks. The type I interferon pathway is detrimental to the vasculature and may contribute to the development of insulin resistance. The level of low-density granulocytes, a distinct subset of proinflammatory neutrophils present in SLE, was independently associated with coronary plaque burden and endothelial dysfunction. Invariant natural killer T cells may promote an atheroprotective effect in SLE patients with asymptomatic atherosclerotic plaques. Oxidized lupus high-density lipoprotein promotes proinflammatory responses in macrophages. Recent discoveries have further strengthened the critical role of SLE-related immune dysregulation and metabolic disturbances in promoting accelerated CVD. Understanding how these pathogenic factors promote vascular injury may provide better molecular candidates for therapeutic targeting, and ultimately to improve CVD outcomes.

Dysregulated neutrophil responses and neutrophil extracellular trap formation and degradation in PAPA syndrome

ObjectivesPyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome is characterised by flares of sterile arthritis with neutrophil infiltrate and the overproduction of interleukin (IL)-1β. The purpose of this study was...

Abstract 3784: Intraoperative changes of low density granulocyte (LDG) in gastrointestinal cancer surgery

Abstract Background: Low density granulocytes (LDG) have been shown to be increased in peripheral blood of autoimmune disease, severe sepsis and cancer. In this study, we examined the frequency and function of the LDG in patients who received abdominal surgery for gastrointestinal malignancy. Method: In 57 patients who received laparotomy due to the gastrointestinal cancer ( 5 esophageal, 25 gastric , 27 colorectal cancer), peripheral blood was collected before and after operative procedure in operation room. After Ficoll-Hypaque centrifugation, intermediate layer which consists mainly of mononuclear cells was collected and immunestained with mAbs to CD66b, CD45 as well as 7AAD and their expressions were examined with flow cytometer. CD66b(+) CD45(+) cells were defined as LDG and the ratios of LDG in total CD45(+) leukocytes were calculated in 7AAD(-) live cell fraction. Next, CD66b(+) LDG and CD66b(-) mononuclear cells were separated with MACS method, and LDG were cultured and examined for extracellular traps (NETs) formation in vitro. Finally, CD66b(-) mononuclear cells were cultured on anti-CD3 mAb coated plate with or without autologous LDG, and proliferation of T lymphocytes was examined with CFSE dilution method. Results: The ratio of the LDG is low (median= 1.14%, 0.62 ~1.66%, n=57) at a beginning of surgery. In contrast, the ratio was markedly increased (median= 8.10%, 5.91~10.3%, n=57, p<0.001) at the end of surgery. The ratio of postoperative LDG showed significant correlation with operation time and positive trend with intraoperative bleeding. The LDG showed relatively week expression of CD11b and CD66b, but highly expressed in IL8 receptor and CD62L, suggesting immature LDG. The short-term culture of the LDG produced massive SYTOX-positive web like stricture which was characteristic for NETs. The mixture of tumor cells on the LDG culture demonstrated that many tumor cells were trapped in the NETs within 5 min incubation and vigorously grew thereafter. Finally, addition of the LDG on mononuclear cell culture strongly suppressed the anti-CD3 stimulated T cell division. Conclusion: LDG with immature phenotype increase in circulating blood of the patients who underwent gastrointestinal cancer surgery, probably due to the surgical stress. The LDG produce NETs which may efficiently trap circulating tumor cells, and can inhibit T lymphocyte mediated-immune reaction. Thus, the LDG provide tumor permissive microenvironment in target organ and have supportive roles on the development of postoperative hematogenous metastasis in gastrointestinal cancer. Citation Format: Yuko Kumagai. Intraoperative changes of low density granulocyte (LDG) in gastrointestinal cancer surgery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3784.

A gene expression module that identifies low-density granulocytes in the blood of patients with systemic lupus erythematosus is associated with low complement, elevated anti-dsDNA titer and both the interferon and TNF signatures

Abstract SLE is an autoimmune disease characterized by the presence of abnormal low-density granulocytes (LDG) with a heightened capacity for spontaneous NETosis. To characterize LDGs in SLE, gene expression profiles derived from isolated LDGs were characterized by Weighted Gene Co-expression Network Analysis, and a 92 gene module was identified. This module contains 30 genes with the Neutrophil Degranulation GO designation and many genes associated with DNA synthesis and proliferation. Comparison with gene expression by bone marrow neutrophil precursors demonstrated that the module of genes was most similar to that expressed by promyelocytes/myelocytes. The LDG gene signature was assessed in gene expression data sets from SLE whole blood (WB), PBMC, kidney, synovium, and skin. The LDG module was enriched consistently in blood but inconsistently in tissues. Lack of granule protein transcripts in tissues suggests that LDGs are not enriched in SLE affected organs. The LDG gene signature was also analyzed in WB and PBMC of 1,417 SLE patients by Gene Set Variation Analysis (GSVA). 35% of inactive patients and 50% of active patients expressed an LDG signature, which showed a significant but modest correlation with disease activity as measured by the SLE Disease Activity Index (r = 0.16, p = 4.1E-9). The LDG signature was also associated with the presence of anti-dsDNA (p < 1E-6), decreased levels of C3 and C4 (p < 1E-6), the type 1 interferon signature (p < 1E-11) and a module of genes induced by TNF (p < 1E-15, all by Fisher’s exact test). These findings suggest that LDGs are a population of immature neutrophils over-represented in the blood of SLE patients in association with cytokine stimulation, the presence of autoantibodies, and complement consumption.

IL-17A induced hematopoietic reprogramming produces both PMN and MDSC at the post-acute stage of inflammation.

Abstract Our previous study suggests that polymorphonuclear leukocyte (PMN) response is enhanced at the post-acute stage of murine colitis due to changes in bone marrow (BM) myelopoiesis. Herein, we report that hematopoietic reprogramming during the post-acute phase of inflammation produces myeloid derived suppressor cells (MDSC) that co-infiltrate the intestine with PMN. Employing Percoll density gradients, we analyzed dynamic changes to the myeloid compartment by separating BM leukocytes from DSS colitis-to-recovery mice into four density-increasing bands with myeloid leukocytes enriched in bands III (50–60%) and IV (60–70%). Band III was comprised of monocytes and low-density granulocytes; both confirmed to be M-MDSCs and G-MDSCs, respectively, by displaying potent inhibition of T-cell proliferation. Despite significant decreases in the PMN population, presumably caused by mobilization into inflamed tissues, MDSC numbers significantly increase beginning at the post-acute stage of colitis. Furthermore, MDSC numbers do not return to baseline until late in the recovery stage. The expanded G-MDSCs also showed increased CXCR2 expression, which guides egress out of BM, and produced ROS upon PMA activation. Adoptive transfer assays demonstrated that functionally enhanced PMN infiltrated the intestine at the post-acute stage to clear the existing inflammatory reaction, while MDSC accumulated in the intestine during recovery suggesting a role in tissue repair. IL-17A signaling at the post-acute stage of inflammation likely induced hematopoietic reprogramming of the myeloid compartment, as its neutralization blocked expansion and infiltration of both PMN and MDSC.

Soluble LOX-1: A Potential biomarker for SLE and Cardiovascular Comorbidity

Abstract Cardiovascular disease is the leading cause of mortality among systemic lupus erythematosus (SLE) patients. LOX-1 is a scavenger receptor strongly implicated in plaque formation, progression and destabilization. It gets upregulated during vascular inflammation on endothelial cells, monocytes, macrophages, smooth muscle cells and platelets. Soluble LOX-1 (sLOX-1) can serve as a potential biomarker for identifying SLE patients with increased cardiovascular risk. In a 120 SLE patient cohort, circulating sLOX-1 levels were 2-fold higher compared to healthy controls, particularly in those14–40 years of age. Patients with high sLOX-1 had elevated proinflammatory HDL, oxLDL, CRP and SAA levels; known markers for vascular injury. High sLOX-1 patient HDL had higher cholesterol content and lower efflux capacity compared to healthy and low sLOX-1 HDL. Both patient derived monocytes and low-density granulocytes (LDGs) express significant levels of membrane LOX-1 compared to controls. Monocytes when conditioned with oxLDL and stimulated with immune complexes, release significant levels of sLOX-1 and cytokines through the LOX-1 receptor. SLE HDL failed to nuclear colocalize the inflammation resolution factor ATF3, in monocyte derived macrophages. Additionally, patient LDGs underwent rapid NETosis in the presence of immune complexes when primed with oxLDL, through the LOX-1 receptor. Disease modified lipoproteins in SLE function as LOX-1 ligand contributing to inflammatory immune activation adding to cardiovascular risk. Hence, sLOX-1 can be predictive of increased cardiovascular risk and LOX-1 receptor blockade may be a promising target for ameliorating cardiovascular damage in SLE.

Neutrophil extracellular trap release is associated with antinuclear antibodies in systemic lupus erythematosus and anti-phospholipid syndrome.

Increased release of neutrophil extracellular traps (NETs) is implicated in the activation of plasmacytoid dendritic cells, vascular disease and thrombosis in SLE and APS. However, studies comparing NET release between patients with SLE and APS are lacking. Here we evaluated plasma-induced NET release in a large cohort of patients with SLE, SLE + APS and primary APS in relation to clinical and serological parameters. Neutrophils from healthy controls were exposed to plasma of heterologous healthy controls (n = 27) or SLE (n = 55), SLE + APS (n = 38) or primary APS (PAPS) (n = 28) patients and NET release was quantified by immunofluorescence. In a subset of SLE patients, NET release was assessed in longitudinal samples before and after a change in treatment. Plasma-induced NET release was increased in SLE and APS patients, with the highest NET release found in patients with SLE (±APS). Plasma of 60% of SLE, 61% of SLE + APS and 45% of PAPS patients induced NET release. NET release did not correlate with disease activity in SLE or APS. However, increased levels of anti-nuclear and anti-dsDNA autoantibodies were associated with increased NET release in SLE and APS. Only in SLE patients, elevated NET release and an increased number of low-density granulocytes were associated with a high IFN signature. Increased NET release is associated with autoimmunity and inflammation in SLE and APS. Inhibition of NET release thus could be of potential benefit in a subset of patients with SLE and APS.

Differential ubiquitination in NETs regulates macrophage responses in systemic lupus erythematosus

ObjectivesTo assess if ubiquitinated proteins potentially present in neutrophil extracellular traps (NETs) can modify cellular responses and induce inflammatory mechanisms in patients with systemic lupus erythematosus (SLE) and healthy subjects.Materials...

Role of neutrophil dysfunction in the pathogenesis of systemic lupus erythematosus

Neutrophil dysfunction plays a considerable role.in systemic lupus erythematosus (SLE) The protective function of neutrophils is carried out through various mechanisms: isolation of granular antimicrobial peptides (gAMP), microbial phagocytosis with subsequent degradation via reactive oxygen species inside the phagolysosomes; as well as bactericidal action due to the release of networks from chromatin and gAMP, also called neutrophil extracellular traps (NECTs). The development of neutropenia in SLE has multiple causes, including the formation of antibodies directly to leukocytes; that of neutralizing autoantibodies to the growth factors of neutrophils and cells - myeloid precursors; bone marrow suppression; involvement of neutrophils in the processes of apoptosis and NETosis. Neutrophils in SLE are characterized by reduced phagocytic ability and pathological oxidative activity. In SLE, there is a decrease in the ability to remove the products of neutrophil apoptosis, which is correlated with disease activity. SLE patients are noted to have a higher expression level of the genes specific for low-density granulocytes, an abnormal immature neutrophil population. The impaired processes of formation of NECTs and removal NETosis products play a substantial role in the pathogenesis of SLE. It is shown that the abnormal formation of NECTs also causes endothelial injury and increases the risk of thromboses. The design of novel drugs that act on the specific parts of the formation of NECTs or contribute to their removal from the extracellular environment can propel therapy for SLE and other autoimmune diseases to new heights. There is evidence for further investigations of neutrophil-mediated pathogenetic processes in SLE in order to identify potential therapeutic targets and to understand the mechanisms of action of drugs used in clinical practice.

Tumor conditions induce bone marrow expansion of granulocytic, but not monocytic, immunosuppressive leukocytes with increased CXCR2 expression in mice.

Myeloid-derived suppressor cells (MDSCs) promote tumor growth through, in part, inhibiting T-cell immunity. However, mechanisms underlying MDSC expansion and guidance of MDSCs toward the tumor microenvironment remain unclear. Employing Percoll density gradients, we separate bone marrow (BM) leukocytes from tumor-bearing mice into four density-increasing bands with myeloid leukocytes enriched in bands III and IV. Band III comprises monocytes and low-density granulocytes, both confirmed to be M-MDSCs and G-MDSCs, respectively, by displaying potent inhibition of T-cell proliferation. However, monocytes act as M-MDSCs not only under tumor conditions but also the healthy condition. In contrast, band IV contains non-inhibitory, mature granulocytes. Only band III G-MDSCs display significant expansion in mice bearing B16 melanoma, Lewis lung carcinoma, or MC38 colon carcinoma. The expanded G-MDSCs also show increased CXCR2 expression, which guides egress out of BM, and produce arginase-1 and ROS upon encountering antigen-activated T cells. Adoptive transfer assays demonstrate that both G-MDSCs and mature granulocytes infiltrate tumors, but only the former displays sustention and accumulation. Intratumoral administrations of granulocytes further demonstrate that G-MDSCs promote tumor growth, whereas mature granulocytes exert minimal effects, or execute powerful anti-tumor effects providing the presence of PMN activation mechanisms in the tumor microenvironment.

Emerging Associations Between Neutrophils, Atherosclerosis, and Psoriasis.

Atherogenesis, once thought to be a passive process, is now recognized as a dynamic, immune-driven process. The critical innate immune cells, including neutrophils, normal-density granulocytes, and their newly identified subset low-density granulocytes, are moving to the forefront of interest in cardiovascular medicine due to their abundance in atherosclerotic plaques and chronic inflammatory diseases associating with early cardiovascular disease (CVD) such as psoriasis. In this review, we discuss the emerging roles of neutrophils in CVD and how they play a potential role in early CVD observed in psoriasis patients. This review aims to describe the roles of neutrophils in both early atherosclerosis and psoriasis. Recent work has demonstrated mechanistic links between vascular inflammation and neutrophil frequency. Evolving mouse models and clinical trials targeting IL-17-associated pathways continue to elucidate contributions of neutrophils in both atherosclerosis and psoriasis. Early animal, in vitro and human studies suggest an important emerging role of neutrophils in atherosclerosis and psoriasis.

Characterization of Circulating Low-Density Neutrophils Intrinsic Properties in Healthy and Asthmatic Horses

Low-density neutrophils (LDNs) are a subset of neutrophils first described in the bloodstream upon pathological conditions, and recently, in the blood of healthy humans. LDNs may have an enhanced pro-inflammatory (low-density granulocytes, LDGs) or an immunosuppressive (Granulocytic myeloid-derived suppressor cells, G-MDSCs) profile. Whether these characteristics are specific to LDNs or related to disease states is unknown. Thus, we sought to investigate the properties of LDNs in both health and disease states, and to compare them to those of autologous normal-density neutrophils (NDNs). We studied 8 horses with severe equine asthma and 11 healthy animals. LDNs were smaller and contained more N-formylmethionine-leucyl-phenylalanine receptors than NDNs, but the myeloperoxidase content was similar in both cell populations. They also had an increased capacity to produce neutrophil extracellular traps, and were more sensitive to activation by phorbol-12-myristate-13-acetate. This profile is suggestive of LDGs. These characteristics were similar in both healthy and diseased animals, indicating that these are intrinsic properties of LDNs. Furthermore, these results suggest that LDNs represent a population of primed and predominantly mature cells. This study is the first to characterize LDNs in health, and to compare their properties with those of NDNs and of animals with a naturally occurring disease.

A simple method for removing low-density granulocytes to purify T lymphocytes from peripheral blood mononuclear cells.

Low-density granulocytes (LDGs) can form neutrophil extracellular traps (NETs) spontaneously and excessively. When peripheral blood mononuclear cells (PBMCs) are used for studying T lymphocytes, LDGs contained in the PBMCs may decrease the threshold of activating T lymphocytes by forming NETs. This study focused on the profiles of LDGs in common autoimmune diseases and methods for removing LDGs from PBMCs. The percentages of LDGs in PBMCs from 55 patients with dermatomyositis (DM), 15 with polymyositis (PM), 42 with rheumatoid arthritis (RA), 25 with systemic lupus erythematosus (SLE), and 19 healthy controls were determined by flow cytometry. Three methods of removing LDGs were explored and compared. After removal, PBMCs from six patients with positive T-SPOT.TB were tested again to find out if LDGs contained in the PBMCs could influence T lymphocyte reactions. Significantly higher LDG percentages were found in PBMCs from patients with DM ((8.41±10.87)%, P<0.0001), PM ((8.41±10.39)%, P<0.0001), RA ((4.05±6.97)%, P=0.0249), and SLE ((7.53±11.52)%, P=0.0006), compared with the controls ((1.28±0.73)%). The T-SPOT.TB values significantly decreased after LDGs were removed. Increasing relative centrifugal force (RCF) within a limited range can decrease the LDG percentage from an initial high level, but not markedly increase the LDG clearance rate. Compared with the whole blood sediment method, the PBMC adherence method can significantly remove LDGs yet scarcely influence the T lymphocyte percentage in PBMCs. The LDG percentage in PBMCs is significantly increased in patients with SLE, DM, PM, and RA. The influence of LDGs on T lymphocytes cannot be ignored in PBMC cultures. The adherence method is a simple and easy-to-use method for removing LDGs and purifying T lymphocytes from PBMCs.

Abstract 4002: Neutrophil extracellular traps (NETs) in abdominal cavity have supportive roles on postoperative development of peritoneal metastasis

Abstract Background: Peritoneal lavages just after abdominal surgery contain many immune cells which consist mainly of granulocytes. Recent studies have demonstrated that neutrophil extracellular traps(NETs) play important roles not only on capture of pathogenic microbes but also on autoimmune disease, thrombus formation and tumor metastasis. Here, in this study, examined the NET forming capacity of granulocytes in postoperative peritoneal washings and assessed the possible role of the NETs on metastasis formation on peritoneum. Method: In patients who underwent laparotomy due to digestive malignancies, abdominal cavity were washed with 1000 ml of normal saline at the timing of laparotomy (Preoperative) and just before wound closure (Postoperative). Cells recovered from the peritoneal washings were centrifuged on Ficoll-Hypaque solution and cultured at 37c in 5 percentage CO2 condition for 24 hour. Then, extracellular ds-DNA were stained with membrane impermeable dye, SYTOX. Also, the granulocytes were immunostained with mAbs against CD66b, histone, myeloperoxidase (MPO), neutrophil elastase (NE) and Matrix metalloproteinase (MMP)-9, and NETs structures were observed under fluorescein microscopy. In adhesion experiments, human gastric cancer cells were stained by PKH-26 and mixed with the granulocyte culture for 5 min and gently washed. Results: 1. After Ficoll separation, CD66b (+) granulocytes were contained both in polymorophonuclear (PMN) and mononuclear cell (PBMC) layers, which are considered to be normo-density granulocytes (NDG) and low density granulocytes (LDG), respectively. The percentage of LDG in PBMC layer was markedly elevated in postoperative as compared preoperative lavages (Pre: M=0.71%, 0~8.9% vs Post: M=61.3%, 3~86.7% n=39, p&amp;lt;0.001) The LDG in postoperative sample showed multinuclear morphology. 2. The short term culture of the postoperative peritoneal LDG, but not preoperative or peripheral blood granulocytes, many SYTOX (+) fibrous structures were observed, suggesting the vigorous NETs formation. The NETs appeared several min after the incubation, peaked at 2 hour and gradually decreased thereafter. Immunostaining showed that histone and MPO were co-localized on the NETs, while NE or MMP-9 were less clear on NETs. 3. After 5 min co-incubation, human gastric cancer cell, OCUM-1 and MKN45, were selectively attached on the NETs. 4. Similar SYTOX (+) NETs-like structure were clearly detected on the surface of human omental tissue resected by abdominal surgery. Conclusion: After laparotomy, many LDG appear in human abdominal cavity which produce large amounts of NETs on peritoneal surface. The NETs efficiently trap the floating tumor cells, which may have positive roles on peritoneal recurrence in advanced cancer. Citation Format: Rihito Kanamaru, Hidenori Tsukui, Shin Saito, Kentaro Kurashina, Takashi Ui, Shiro Matsumoto, Hironori Yamaguchi, Yasunaru Sakuma, Hisanaga Horie, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. Neutrophil extracellular traps (NETs) in abdominal cavity have supportive roles on postoperative development of peritoneal metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4002. doi:10.1158/1538-7445.AM2017-4002

Neutrophil-to-lymphocyte ratio correlates with proinflammatory neutrophils and predicts death in low model for end-stage liver disease patients with cirrhosis.

The Model for End-Stage Liver Disease (MELD) score has reduced accuracy for liver transplantation (LT) wait-list mortality when MELD ≤ 20. Neutrophil-to-lymphocyte ratio (NLR) is a biomarker associated with systemic inflammation and may predict cirrhotic decompensation and death. We aimed to evaluate the prognostic utility of high NLR (≥4) for liver-related death among low MELD patients listed for LT, controlling for stage of cirrhosis. In a nested case-control study of cirrhotic adults awaiting LT (February 2002 to May 2011), cases were LT candidates with a liver-related death and MELD ≤ 20 within 90 days of death. Controls were similar LT candidates who were alive for ≥90 days after LT listing. NLR and other covariates were assessed at the date of lowest MELD, within 90 days of death for cases and within 90 days after listing for controls. There were 41 cases and 66 controls; MELD scores were similar. NLR 25th, 50th, 75th percentile cutoffs were 1.9, 3.1, and 6.8. NLR was ≥ 4 in 25/41 (61%) cases and in 17/66 (26%) controls. In univariate analysis, NLR (continuous ≥ 1.9, ≥ 4, ≥ 6.8), increasing cirrhosis stage, jaundice, encephalopathy, serum sodium, and albumin and nonselective beta-blocker use were significantly (P < 0.01) associated with liver-related death. In multivariate analysis, NLR of ≥1.9, ≥ 4, ≥ 6.8 were each associated with liver-related death. Furthermore, we found that NLR correlated with the frequency of circulating low-density granulocytes, previously identified as displaying proinflammatory properties, as well as monocytes. In conclusion, elevated NLR is associated with liver-related death, independent of MELD and cirrhosis stage. High NLR may aid in determining risk for cirrhotic decompensation, need for increased monitoring, and urgency for expedited LT in candidates with low MELD. Liver Transplantation 23 155-165 2017 AASLD.

Tocilizumab improves the proatherothrombotic profile of rheumatoid arthritis patients modulating endothelial dysfunction, NETosis, and inflammation

Tocilizumab (TCZ) is an effective treatment for rheumatoid arthritis (RA). However, the changes that occurred after TCZ therapy on endothelial dysfunction, monocyte activity, NETosis, and oxidative stress, the principal effectors of atherosclerosis and cardiovascular disease, have not been analyzed yet. A total of 20 RA patients received 162mg per week subcutaneous TCZ for 6months. Endothelial function was measured through postocclusive hyperemia using Laser Doppler. Oxidative stress markers in monocytes and neutrophils were analyzed by flow cytometry. NETosis was measured through SYTOX staining of DNA fibers and the expression of myeloperoxidase and neutrophil elastase. Percentage of low-density granulocytes was analyzed through flow cytometry. Gene expression and phosphorylation of intracellular pathways was analyzed in monocytes. TCZ improved endothelial function and decreased oxidative stress in RA leukocytes. Percentage of low-density granulocytes and NETosis generation were reduced. The proinflammatory and prothrombotic status of RA monocytes was also reversed through a modulation of specific intracellular pathways. All these results were recapitulated after invitro treatment with TCZ of monocytes and neutrophils purified from RA patients and cocultured with endothelial cells. TCZ might reduce the proatherothrombotic profile in RA patients through the restoration of the endothelial function, oxidative stress reduction, inhibition of monocytes' prothrombotic and inflammatory profile, and abridged NETosis generation.

G-CSF-Induced Suppressor IL-10+ Neutrophils Promote Regulatory T Cells That Inhibit Graft-Versus-Host Disease in a Long-Lasting and Specific Way.

Acute graft-versus-host disease (aGVHD) is the main complication of allogeneic hematopoietic stem cell transplantation, and many efforts have been made to overcome this important limitation. We showed previously that G-CSF treatment generates low-density splenic granulocytes that inhibit experimental aGVHD. In this article, we show that aGVHD protection relies on incoming IL-10+ neutrophils from G-CSF-treated donor spleen (G-Neutrophils). These G-Neutrophils have high phagocytic capacity, high peroxide production, low myeloperoxidase activity, and low cytoplasmic granule content, which accounts for their low density. Furthermore, they have low expression of MHC class II, costimulatory molecules, and low arginase1 expression. Also, they have low IFN-γ, IL-17F, IL-2, and IL-12 levels, with increased IL-10 production and NO synthase 2 expression. These features are in accordance with the modulatory capacity of G-Neutrophils on regulatory T cell (Treg) generation. In vivo, CD25+ Treg depletion shortly after transplantation with splenic cells from G-CSF-treated donors blocks suppression of aGVHD, suggesting Treg involvement in the protection induced by the G-Neutrophils. The immunocompetence and specificity of the semiallogeneic T cells, long-term after the bone marrow transplant using G-Neutrophils, were confirmed by third-party skin graft rejection; importantly, a graft-versus-leukemia assay showed that T cell activity was maintained, and all of the leukemic cells were eliminated. We conclude that G-CSF treatment generates a population of activated and suppressive G-Neutrophils that reduces aGVHD in an IL-10- and Treg-dependent manner, while maintaining immunocompetence and the graft versus leukemia effect.

Low-density granulocytes: functionally distinct, immature neutrophils in rheumatoid arthritis with altered properties and defective TNF signalling.

Our aim was to determine whether rheumatoid arthritis (RA) low-density granulocytes (LDGs) are functionally different from RA neutrophils. LDGs from 32 RA patients were characterized using flow cytometry and quantitative PCR (qPCR). RNA sequencing (RNA-Seq) was carried out on paired RA LDGs and neutrophils (n = 4) and validated using qPCR. Functional assays included chemotaxis, phagocytosis, reactive oxygen species (ROS) production, cell-cycle analysis, apoptosis, neutrophil extracellular trap (NET)osis, and measurement of cytokine production (n ≥ 5 paired RA LDGs/neutrophils). RA LDGs had a substantially altered transcriptome, expressing >5000 genes at significantly different levels compared with RA neutrophils, including elevated levels of transcripts for granule proteins [including elastase and myeloperoxidase (MPO)] and cell-cycle genes [including cyclin-dependent kinase (CDK)2, CDK4, and CDK6]. Approximately 1% of RA LDGs stained positive for the G2/S phase of the cell cycle. RA LDGs had a significantly lower constitutive rate of apoptosis compared with RA neutrophils and did not respond to TNF-α in culture. Expression of transcripts for cytokines and cytokine receptors was lower in RA LDGs. NET formation was lower in LDGs in response to PMA compared with RA neutrophils. Chemotaxis and phagocytosis was lower in RA LDGs compared with neutrophils. RA LDGs produced significantly lower amounts of ROS in response to fMLP following priming with TNF-α. Expression of TNFR1 and -2 mRNA and protein was significantly lower in LDGs. We conclude that RA LDGS are functionally different from RA neutrophils, representing an immature neutrophil population within peripheral blood. Their enhanced survival properties and decreased TNF signaling are likely to have important consequences for disease pathology and response to therapy.

Human neutrophils in the saga of cellular heterogeneity: insights and open questions.

Recent findings have uncovered novel fascinating aspects of the biology of neutrophils, which ultimately attribute to these cells a broader role in inflammation and immunity. One aspect that is currently under intensive investigation is the notion of neutrophil 'heterogeneity'. Studies examining neutrophils in a variety of acute and chronic inflammatory conditions report, in fact, the recovery of CD66b(+) cells displaying neutrophil-like morphology at different degrees of maturation/activation, able to exert either immunosuppressive or proinflammatory properties. These heterogeneous populations of mature and immature neutrophils are indicated with a variety of names, including 'low density neutrophils (LDNs)', 'low density granulocytes (LDGs)', 'granulocytic-myeloid derived suppressor cells (G-MDSCs)', and immunosuppressive neutrophils. However, due to the lack of discrete markers that can unequivocally allow their specific identification and isolation, the precise phenotype and function of all these presumably novel, neutrophil-like, populations have not been correctly defined yet. Aim of this article is to summarize current knowledge on the mature and immature neutrophil populations described to date, featuring immunosuppressive or proinflammatory properties, often defined as 'subsets', as well as to critically discuss unresolved issues in the field.

Abnormally increased low-density granulocytes in peripheral blood mononuclear cells are associated with interstitial lung disease in dermatomyositis

Objective: We previously found that neutrophil extracellular traps (NETs) were associated with interstitial lung disease (ILD) in dermatomyositis (DM) patients. However, it is unclear whether low-density granulocytes (LDGs), endowed with enhanced NET formation capabilities, contribute to the pathogenesis of ILD. This study aims to elucidate the relationship between LDGs and DM-associated ILD.Methods: We recruited 48 DM patients (28 with ILD) as well as 19 healthy volunteers for this study. The percentage of LDGs in peripheral blood mononuclear cells (PBMCs) was ascertained by flow cytometry. Plasma cfDNA was measured by using the Quant-iT PicoGreen dsDNA Kit and plasma LL-37 was tested by using the LL-37 ELISA kit.Results: The percentage of LDGs was 7.1 times higher in DM patients than in healthy controls. LDG percentage was 2.7 times higher in DM patients with ILD than in DM patients without ILD. Additionally, LDG percentage positively correlated with MYOACT lung disease activity scores, and NET/neutrophil-related marker levels (LL-37, cfDNA, MPO, and MMP-8) in the DM group were significantly higher than those in the control group.Conclusion: The abnormal increase of LDGs may exacerbate abnormal NET regulation and further contribute to the pathogenesis of ILD in DM patients by abnormally forming NETs.